The major peroxidase of barley grain (BP 1) has enzymatic and spectroscopic properties that are very differeant from those of other known plant peroxidases (EC 22.214.171.124) and can therefore contribute to the understanding of the many physiological functions ascribed to these enzymes. To study the structure-function relationships of this unique model peroxidase, large-scale and laboratory-scale purifications have been developed. The two batches of pure BP 1 obtained were identical in their enzymatic and spectral properties, and confirmed that BP 1 is different from the prototypical horseradish peroxidase isoenzyme C (HRP C). However, when measuring the specific activity of BP 1 at pH 4.0 in the presence of 1 mM CaCl2, the enzyme was as competent as HRP C at neutral pH towards a variety of substrates (mM mg(-1) min(-1)): coniferyl alcohol (930+/-48), caffeic acid (795+/-53), ABTS (2,2(1)-azino-di-[3-ethyl-benzothiazoline-(6) sulfonic acid]) (840+/-47), ferulic acid (415+/-20), p-coumaric acid (325+/-12), and guaiacol (58+/-3). The absorption spectrum of BP 1 is blue-shifted compared to that of HRP C with a Soret maximum of 399-402 nm, depending on pH. The prosthetic group was shown to be iron-protoporphyrin IX, which is characteristic of plant peroxidases. BP 1 is stable from pH 3 to 11, indicating that its unusual spectral characteristics do not result from enzyme instability. The thermostability is also normal with a melting temperature of 75 degrees C at pH 6.6, and 67 degrees C at pH 4.0 and 8.3. It is clear that the unusual properties of BP 1 are genuine, and reflect a novel regulation of plant peroxidase function.
|Publication status||Published - 1997|